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Abstract:

The present invention provides compositions and methods of use thereof to
prevent and/or treat pathogenic infection. In particular, the present
invention provides the use of kinase inhibitors to inhibit kinases that
involve in pathogen-host cell interactions that are associated with or
cause pathogenic infections, therefore, to effectively prevent and/or
treat pathogenic infections with far less likely to engender resistance
as compared to conventional antibiotics and anti-viral drugs. The present
invention further provides the use of kinase inhibitors for the treatment
of acute pathogenic infections for a short period of time to avoid
toxicities that may caused by long term use of these kinase inhibitors.

Claims:

1. A method of preventing or treating pathogenic infection comprising
administering a therapeutically effective amount of compositions
comprising one or more kinase inhibitors as set forth in Table A to a
patient in need thereof for preventing or treating infection caused by a
broad array of pathogens.

13. The method of claim 1, wherein said pathogenic infection is an acute
infection.

14. The method of claim 13, wherein said acute infection is treated for
short periods of time.

15. The method of claim 14, wherein said short periods of time is less
than three weeks.

16. The method of claim 1, wherein said kinase inhibitors are set forth
in Summary Table B.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of U.S. application Ser. No.
12/439,961, filed Oct. 1, 2009, which is a U.S. national stage filing of
PCT/US2007/077578, filed Sep. 5, 2007, which claims benefit of U.S.
Provisional Application No. 60/824,540, filed Sep. 5, 2006. The contents
of the above-referenced applications are incorporated herein by reference
in their entirety.

FIELD OF THE INVENTION

[0003] The invention relates to compositions and methods of use thereof to
prevent and/or treat pathogenic infection. In particular, the present
invention relates to a development and identification of compounds that
alter the way in which diverse bacterial and viral pathogens interact
with the host, so as to block or limit disease caused by these pathogens
and permit the host immune system to clear the pathogens.

BACKGROUND OF THE INVENTION

[0004] The last several decades have witnessed an onslaught of deadly
bacterial and viral pathogens around the globe. A broad array of human
pathogens exists, including various microbes such as bacteria, protozoa,
viruses, algae, and fungi. The innate capacity to respond to selective
pressures has driven the evolution of microbes and enabled them to adapt
to complex and variable environments. It is perhaps no surprise, then,
that infectious microbes have readily evolved mechanisms to evade our
attempts to destroy them with synthetic or natural anti-microbial
compounds.

[0005] The fact that microbes develop resistance at a rate that far
exceeds development of new therapeutics arguably poses the single most
serious public health threat in this century in both developing and
developed nations. There is no denying that anti-microbial strategies
have met with spectacular success over the last century.

[0006] For example, antibacterial and antiviral drugs directed at targets
within the pathogen have been used to save countless lives. But it is
becoming increasingly evident that such success is not sustainable. To
counter these drugs, bacterial and viral pathogens have evolved
sophisticated mechanisms to inactivate these compounds. Examples include
the pan-drug resistant strains of Staphylococcus aureus, Klebsiella
pneumoniae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis (TB)
among bacteria and human immunodeficiency virus (HIV) among viruses.

[0007] More worrisome still is the lack of effort on the part of
pharmaceutical companies (big or small) to pursue development of new
antimicrobials. Efforts to develop new antibiotics by the pharmaceutical
industry by large-scale screens of chemical libraries that inhibit growth
have largely failed, and new tetracycline and sulfanilamide analogs will
likely engender resistance and will quickly be rendered useless. The
resistance problem is compounded further by indiscriminate and
inappropriate use of antibiotics and antiviral compounds without
compliance measures or public health policies to reduce disease burden.
With the astounding costs of clinical trials, the failure to control
generic sales, and the capacity to generate substantial revenues from
medications for chronic illnesses there is little if any financial
incentive for big pharmaceutical companies to even develop new
antibiotics, and small biotechnology companies simply do not have the
resources.

[0008] Even with the current level of effort there is cause for concern.
Of the new drugs under development, most, if not all, will likely
engender resistance quickly upon release (e.g., folate biosynthesis
inhibitor Iclaprim). The search for novel antiviral compounds has been
somewhat more successful and largely motivated by the HIV pandemic, but
drugs have been developed principally against viral targets, and mutation
rates among viruses still outpaces new development. One positive
development has been vaccines, which are promising for some bacterial and
viral illnesses. But vaccines are not successful in all cases (e.g., in
young children), and adequate resources have not been made available.

[0009] There is therefore an urgent need to develop compounds and methods
effective for the prevention and treatment of pathogenic infection.

SUMMARY OF THE INVENTION

[0010] The present invention provides compounds that alter the way in
which diverse bacterial and viral pathogens interact with the host. The
compounds provided by the present invention interact with host proteins
required by microbes for pathogenesis. As such, the compounds provided by
the present invention are far less likely to engender resistance compared
to conventional antibiotics or anti-viral drugs because the pathogen
cannot easily evolve novel pathogenesis strategies. Therefore, the
compounds provided by the present invention have the capacity to limit
disease and permit the host immune system to clear the pathogen. In one
preferred embodiment, the present invention provides compounds that
inhibit kinases involved in pathogen-host cell interactions that are
associated with or cause pathogenic infection. The kinase inhibitors of
the present invention include, but are not limited, to the compounds
listed in Table A below. In yet another preferred embodiment, the kinase
inhibitors of the present invention are used for the treatment of acute
pathogenic infections for a short period of time, preferably, less than 3
weeks, to avoid toxicity issues.

[0012] In yet another preferred embodiment, the present invention provides
compositions comprising compounds including those listed in Table A below
that inhibit kinases involved in pathogen-host cell interactions that are
associated with or cause pathogenic infection. In one of the preferred
embodiments, the kinase is tyrosine kinase. In yet another preferred
embodiment, the present invention provides compositions comprising
inhibitors to tyrosine kinase, preferably, Ableson (Abl) and/or
Src-family tyrosine kinase, or pharmaceutically acceptable salts,
enantiomers, analogs, esters, amides, prodrugs, metabolites, or
derivatives thereof.

[0013] In yet another preferred embodiment, the present invention provides
methods of preventing or treating pathogenic infections. Such methods
comprise administering the compositions comprising kinase inhibitors of
the present invention in therapeutically effective amounts to a patient
in need thereof for treating infection by a broad array of pathogens,
including microbial pathogens such as bacteria, protozoa, viruses, algae,
and fungi. In particular, the present invention provides the use of these
compositions to treat disease associated with the pathogens including
Escherichia coli (enteropathogenic Escherichia coli (EPEC),
enterohemorrhagic Escherichia coli (EHEC), uropathogenic Escherichia coli
(UPEC), and enteroinvasive Escherichia coli (EIEC)), Mycobacterium
tuberculosis (mTB), Pseudomonas aeruginosa, Chlamydia trachomatis, Pox
viruses (including Vaccinia and variola viruses), polyoma viruses
(including JC and BK viruses), human immunodeficiency viruses (for
example, HIV-1), Herpes viruses (including Herpes Simplex virus, Epstein
Barr virus, and Gamma Herpes virus), influenza virus, Shigella flexneri,
Coxsackie virus, Helicobacter pylori, West Nile virus, Listeria
monocytogenes, Salmonella typhimurium, cytomegalovirus (CMV), and other
pathogens that are described in the literature. In one of the preferred
embodiments, the present invention provides the use of these compositions
to treat acute pathogenic infections for a short period of time,
preferably, less than three weeks, to avoid toxicity. The compositions
may be administered by any means of administration as long as a
therapeutically effective amount for the treatment of pathogenic
infection is delivered.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS. 1A-C illustrate small plaque formations due to drug treatment
in Plaque Assays. FIG. 1A shows plaque formation with vaccinia virus in
the absence of any kinase inhibitors in 3T3 cells, strain WR (left:
Positive Control), and in the absence of virus and any kinase inhibitors
(Right: Negative Control); FIG. 1B shows formations of small plaques with
comets with compounds Eph--2wbz--105, Eph--2wbz--203,
Eph--2wbz--206 and LG2-71, respectively; and FIG. 1C shows
formations of small plaques with no comets with compounds DM-1-187 and
DM-1-196, respectively.

[0018] The present invention provides compositions comprising compounds
that inhibit kinases involved in pathogen-host cell interactions that are
associated with or cause pathogenic infection and methods of using such
compositions. The compounds of the present invention include, but are not
limited to those listed in the following Table A. As used herein, the
terms "compounds" and "kinase inhibitors" are used interchangeably,
referring to chemicals that are capable of interacting with kinases
involved in pathogen-host cell interactions that are associated with or
cause pathogen infections, including but not limited to those chemicals
with the structures shown in the following Table A.

[0019] One type of the kinase inhibitors listed above are inhibitors for
tyrosine kinase that are involved in pathogen-host cell interactions
associated with or cause pathogenic infection. It has been reported that
diverse pathogens activate tyrosine kinases, particularly members of the
Abl- and Src-families. Because Abl- and Src-family kinases are essential
for the host, therapeutics must be dosed properly to minimize spread of
the pathogen without harming the host. Because of the diverse numbers of
pathogens that use Abl- and Src-family kinases (Reeves et al., 2005, Nat.
Med 11: 731-738), the development of "pan-therapeutics" that affect
multiple pathogens is possible. Administration of tyrosine kinase
inhibitors does not appear to interfere with acquisition of protective
immunity (e.g. to poxviruses). Thus administration of therapeutics need
continue only until an effective immune response has been mounted.
Toxicity data from some tyrosine kinase inhibitors in cancer patients
suggest that acute infections, where therapeutics could be administered
for short periods of time (e.g. less than three weeks), would be ideal
targets (Kerkela et al., 2006, Nat. Med. 12(8):908-16).

[0020] Diverse pathogens use kinases in a redundant fashion. Rather than
utilize a single kinase pathway, pathogens appear to have developed
molecular means to utilize several kinases within different subfamilies,
perhaps as a means to increase their host range. Redundancy adds an
element of complexity to the development of therapeutics. Because kinases
that diverse pathogens utilize are dysregulated in a variety of human
cancers, considerable effort has been made over the last two decades in
developing compounds that inhibit these kinase activities. An inhibitor
must be sufficiently non-specific to inhibit the class of kinases used by
the pathogen, but within limits. Current efforts are also directed at
identification of microbial and host molecules phosphorylated by kinases.
Such molecules are also effective as therapeutic targets. It has also
been found that some anti-cancer drugs have proven effective against a
variety of pathogens (Reeves et al., 2005, Nat. Med 11: 731-738).

[0022] The kinase inhibitors described herein can be used in the methods
of the invention to treat or prevent any pathogenic infection that is
associated with or caused by these kinase-mediated host-pathogen
interactions, particularly microbial infection, and more particularly
viral and bacterial infection. Without being bound by theory, it is
believed that the kinase inhibitors described herein target host cell
proteins and interfere with cellular mechanisms required for pathogenesis
of the host cells by pathogens and in so doing prevent the pathogenic
effects. Because cellular mechanisms regulating pathogen-host
interactions are remarkably conserved, it is believed that the kinase
inhibitors described herein can be applied to combat infection by a wide
range of pathogens. Such pathogens include various microbes such as
bacteria, protozoa, viruses, algae, and fungi. In a preferred embodiment
of the present invention, the pathogens are bacteria and viruses.
Advantageously, the therapeutic approach described herein targets the
host, rather than the pathogen as is seen with antibiotics, and therefore
decreases the likelihood of the development of pathogen drug resistance.

[0024] In another embodiment, the present invention provides the use of
kinase inhibitors of the present invention to treat or prevent viral
infections. Such infections include those caused by members of the
following virus families: Adenoviridae, Arenaviridae, Astroviridae,
Bacteriophages, Baculoviridae, Bunyaviridae, Caliciviridae,
Coronaviridae, Deltavirus, Filoviridae, Flaviviridae, Geminiviridae,
Hepadnaviridae, Herpesviridae, Nodaviridae, Orthomyxoviridae,
Papovaviridae, Paramyxoviridae, Parvoviridae, Phycodnaviridae,
Picornaviridae, Poxyiridae, Reoviridae, Retroviridae, Rhabdoviridae,
Tobamoviridae, and Togaviridae. In a preferred embodiment, such
infections include those caused by Pox viruses including Vaccinia and
variola viruses, polyoma viruses including JC and BK viruses, Herpes
viruses, cytomegalovirus (CMV), and human immunodeficiency viruses (for
example, HIV-1).

[0025] In accordance with the methods of the present invention, the kinase
inhibitors of the present invention described herein may be administered
in combination with one another, or with other compounds, particularly
antipathogenic compounds. Such antipathogenic compounds include
conventional antimicrobials. In other embodiments, one or more of the
kinase inhibitors of the present invention described herein can be used
in combination with other compounds such as cidofovir, for example, in
cases related to smallpox, wherein the combination of these agents would
provide for lower dosages of cidofovir to be administered, thereby
decreasing the toxicity effects of this nucleoside analogue antiviral
compound. Where the kinase inhibitors of the present invention are
administered as part of a combination therapy to treat or prevent
pathogenic infection, they may be administered concurrently or
sequentially, in either order, with the additional compound(s).

[0026] In one embodiment, kinase inhibitors are administered to make
vaccines more effective. For example, it is well known that immunization
of neonates with live viruses does not contribute to acquired immunity
because maternal antibodies neutralize the vaccine (Bot and Bona (2002)
Microbes Infect. 4: 511). In one embodiment, administration of a kinase
inhibitor of the present invention allows for safe administration of
higher doses of virus to overcome antibody response and permit
acquisition of cellular immunity. In another embodiment, kinase
inhibitors of the present invention facilitate immune clearance of the
pathogen. For some chronic viruses (e.g., HIV and polyoma), high viral
loads have been found to compromise T cell function (Welsh (2001) J. Exp.
Med. 193:F19). Thus, lowering the viral burden could permit recovery of T
cell function and thereby facilitate clearance. In another embodiment,
kinase inhibitors of the present invention permit immunocompromised
individuals to be vaccinated.

[0027] The kinase inhibitors of the present invention are for
administration in a living subject or patient, including a human being or
an animal such as a laboratory monkey or mouse. It is to be understood
that the present invention encompasses the use not only of the specific
compounds described above, but also any pharmaceutically acceptable
salts, enantiomers, analogs, esters, amides, prodrugs, metabolites, or
derivatives thereof. Because some of the kinase inhibitors of the present
invention are already the subject of drug development or are in use to
treat certain cancers, data has established that they are well tolerated
in humans even for extended periods (months), and are not toxic. The
drugs can be ingested orally, are stable at room temperature, and are
simple and inexpensive to manufacture.

[0028] In one embodiment of the present invention, a method of treating or
preventing pathogenic infection, particularly microbial infection,
comprises administering to a living subject in need of such treatment an
effective amount of a pharmaceutical composition suitable for
administration to the living subject where the pharmaceutical composition
comprises: (a) at least one kinase inhibitor of the present invention in
an amount effective for augmenting an inhibitable response from a host
cell of the living subject responsive to at least one pathogen,
particularly a microbe; and (b) a pharmaceutically acceptable carrier
suitable for administration to the living subject. In another embodiment,
the present invention provides pharmaceutical compositions suitable for
administration to a living subject, comprising: (a) at least one kinase
inhibitor in an amount effective for augmenting an inhibitable response
from a host cell of the living subject responsive to at least one
bacteria; and (b) a pharmaceutically acceptable carrier suitable for
administration to a living subject. In another embodiment, the present
invention provides pharmaceutical compositions suitable for
administration to a living subject, comprising: (a) at least one kinase
inhibitor in an amount effective for augmenting an inhibitable response
from a host cell of the living subject responsive to at least one virus;
and (b) a pharmaceutically acceptable carrier suitable for administration
to a living subject. In yet another preferred embodiment, the kinase
inhibitors of the present invention are tyrosine kinase inhibitors,
preferably, the Abl- and/or Src-family tyrosine kinase inhibitors.

[0029] Depending upon the pathogenic infection to be treated or prevented,
the pharmaceutical composition comprising a kinase inhibitor of the
present invention described herein can be administered by any suitable
route, including, but not limited to, orally, nasally, buccally,
sublingually, intravenously, transmucosally, rectally, topically,
transdermally, subcutaneously, by inhalation, or intrathecally
administration.

[0030] In one of the preferred embodiments, these pharmaceutical
compositions may be in the form of orally administrable suspensions,
drinking solutions, or tablets; nasal sprays or nasal drops; or
oleaginous suspensions or suppositories. When administered orally as a
suspension, compositions of the present invention are prepared according
to techniques well known in the art of pharmaceutical formulation and may
contain microcrystalline cellulose for imparting bulk, alginic acid or
sodium alginate as a suspending agent, methylcellulose as a viscosity
enhancer, and sweeteners/flavoring agents known in the art. As immediate
release tablets, these compositions may contain microcrystalline
cellulose, dicalcium phosphate, starch, magnesium stearate and lactose
and/or other excipients, binders, extenders, disintegrants, diluents and
lubricants known in the art. Components in the formulation of a mouthwash
or rinse include antimicrobials, surfactants, cosurfactants, oils, water
and other additives such as sweeteners/flavoring agents known in the art.
When administered by a dribbling solution, the composition comprises one
or more of the kinase inhibitors of the present invention described
herein dissolved in drinking liquid such as water, with appropriate pH
adjustment, and with carrier. The compound dissolved in the drinking
liquid is an amount sufficient to give a concentration in the bloodstream
on the order of 1 nM and above, preferably in an effective amount that is
effective in vivo.

[0031] When administered nasally, these compositions are prepared
according to techniques well known in the art of pharmaceutical
formulation and may be prepared as solutions in saline, employing benzyl
alcohol or other suitable preservatives, absorption promoters to enhance
bioavailability, and/or other solubilizing or dispersing agents known in
the art (see, for example, Ansel et al. (1999) Pharmaceutical Dosage
Forms and Drug Delivery Systems (7th ed.). Preferably these compositions
and formulations are prepared with suitable nontoxic pharmaceutically
acceptable ingredients. These ingredients are known to those skilled in
the preparation of nasal dosage forms and some of these can be found in
Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Company,
Eaton, Pa.; 1990), a standard reference in the field. The choice of
suitable carriers is highly dependent upon the exact nature of the nasal
dosage form desired, e.g., solutions, suspensions, ointments, or gels.
Nasal dosage forms generally contain large amounts of water in addition
to the active ingredient. Minor amounts of other ingredients such as pH
adjusters, emulsifiers or dispersing agents, preservatives, surfactants,
gelling agents, or buffering and other stabilizing and solubilizing
agents may also be present.

[0032] The formulations for the kinase inhibitors of the present invention
may be varied to include: (1) other acids and bases to adjust the pH, (2)
other tonicity-imparting agents such as sorbitol, glycerin, and dextrose;
(3) other antimicrobial preservatives such as other parahydroxy benzoic
acid esters, sorbate, benzoate, propionate, chlorbutanol, phenylethyl
alcohol, benzalkonium chloride, and mercurials; (4) other viscosity
imparting agents such as sodium carboxymethylcellulose, microcrystalline
cellulose, polyvinylpyrrolidone, polyvinyl alcohol and other gums; (5)
suitable absorption enhancers; (6) stabilizing agents such as
antioxidants, like bisulfate and ascorbate, metal chelating agents such
as sodium edentate, and drug solubility enhancers such as polyethylene
glycols.

[0033] The above nasal formulations can be administered as drops, sprays,
or by any other intranasal dosage form. Optionally, the delivery system
can be a unit dose delivery system. The volume of solution or suspension
delivered per dose can be anywhere from 5 to 500 microliters, and
preferably 5 to 200 microliters. Delivery systems for these various
dosage forms can be dropper bottles, plastic squeeze units, atomizers,
and the like in either unit dose or multiple dose packages. Lozenges can
be prepared according to U.S. Pat. No. 3,439,089, herein incorporated by
reference for these purposes.

[0034] When rectally administered in the form of suppositories, these
compositions may be prepared by mixing the kinase inhibitors of the
present invention with a suitable non-irritating excipient, such as cocoa
butter, synthetic glyceride esters, or polyethylene glycols, which are
solid at ordinary temperatures, but liquify and/or dissolve in the rectal
cavity to release the drug.

[0035] Dosage levels on the order of 1 mg/day or above may be useful in
the treatment or prevention of pathogenic infections and related diseases
within a host organism as noted herein above. In one embodiment of the
present invention, a patient in need of treatment or prevention of
pathogenic infection is administered a pharmaceutical composition
comprising one or more kinase inhibitors of the present invention
described herein in an effective amount of about 1 mg/day to about 1000
mg/day, for a patient having approximately 70 kg body weight. It will be
understood, however, that the specific dose level and frequency of dosage
for any particular patient may be varied and will depend upon a variety
of factors including the activity of the specific salt or other form
employed, the metabolic stability and length of action of that compound,
the age, body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, the severity of the
particular condition, and the host undergoing therapy. In one preferred
regimen, such dosages can be administered to a subject in need thereof by
either nasal spray or by oral lozenge.

[0036] The effectiveness of using the pharmaceutical compositions of the
present invention to treat or prevent a specific pathogenic infection,
particularly microbial infection, may vary, for example, depending on the
infectious agent, stage of infection, severity of infection, age, weight,
and sex of the patient, and the like.

[0037] As used herein, the term "treatment" is defined as the application
or administration of one or more kinase inhibitors of the present
invention described herein to a subject, where the subject has a
pathogenic infection as noted elsewhere herein, a symptom associated with
a pathogenic infection, or a predisposition toward development of a
pathogenic infection, where the purpose is to cure, heal, alleviate,
relieve, alter, remedy, ameliorate, improve, or affect the pathogenic
infection, any associated symptoms of the pathogenic infection, or the
predisposition toward the development of the pathogenic infection. The
term "treatment" is also defined as an intended application or
administration of a pharmaceutical composition comprising one or more
kinase inhibitors of the present invention described herein to a subject,
where the subject has a pathogenic infection as noted elsewhere herein, a
symptom associated with a pathogenic infection, or a predisposition
toward development of a pathogenic infection, where the purpose is to
cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or
affect the pathogenic infection, any associated symptoms of the
pathogenic infection, or the predisposition toward the development of the
pathogenic infection.

[0038] The kinase inhibitors, particularly the tyrosine kinase inhibitors,
of the present invention described herein are useful in treating or
preventing pathogenic infections as noted herein above. Treatment or
prevention of pathogenic infection in the manner set forth herein is also
useful for transplant patients, for example, kidney transplant patients,
where emergence of pathogens, particularly polyoma viruses, for example,
JC and BK, and pathogenic infection can diminish function of the
transplanted organ. In like manner, HIV infection can destroy
oligodendrocytes in the brain, leading to AIDS-related dementia. Thus, in
addition to treating or preventing pathogenic infections as noted
elsewhere herein, the kinase inhibitors, particularly the tyrosine kinase
inhibitors, of the present invention described herein can be used to
control secondary infection in HIV-positive and AIDS patients and in
patients receiving transplants, for example, kidney transplants, and to
control AIDS-related dementia. Further, the kinase inhibitors,
particularly, the tyrosine kinase inhibitors, can be used
prophylactically to prevent spread of infectious virions, for example,
associated with Vaccinia infections, in immunocompromised individuals,
including HIV-positive and AIDS patients and in patients receiving
transplants.

[0046] It is known that the protein encoded by the VV A36R gene (called
A36R), located in the membrane surrounding the CEV, is required for actin
polymerization; and virulence (Wolffe et al. (1998) Virology pp. 20-26;
Parkinson and Smith (1994) Virology pp. 376-390). The watershed event in
actin polymerization and cell-to-cell spread is the phosphorylation of
A36R tyrosine residues by a host cell tyrosine kinase (Newsome et al.
(2004) Science 306:124-128; Frischknecht et al. (1999) Nature
401(6756):926-929). There is a remarkable homology between the EPEC Tir
protein described above and the VV protein A36R, therefore using similar
but not identical host signaling factors as EPEC to polymerize actin and
exit from the host cell (Frischknecht and Way (2001) Trends Cell Biol.
11(1):30-38).

[0048] These and many other variations and embodiments of the invention
will be apparent to one of skill in the art upon a review of the appended
description and examples.

EXAMPLES

Example 1

Drug Screening Using Microscopy Assays

[0049] The present invention provides drug screening assays for microbal
pathogens. In one of the preferred embodiments, the present invention
provides drug screening assays for viral pathogens, preferably, the
poxviruses. Two exemplary drug screening assays: the microscopy assay and
the Plaque Assay, are provided herein. The purpose of microscopy assays
is to screen compounds in a high throughput format for their effects on
the formation of actin protein filled membranous protrusions caused by
vaccinia virus egressing from an infected cell (or "tails"). The
microscopy assays also reveal, albeit indirectly effects on replication
or viral maturation.

[0050] To do the microscopy assays, cultured 3T3 cells were added at a low
density to collagen/PDL-coated glass microscopy slips or on 96 well
optical tissue culture plates. The cells were allowed to adhere to these
slips overnight. The next day, the media was removed from these cells and
replaced with low-serum media. Approximately 106 vaccinia virus
virions were added directly to the low-serum media and infection was
allowed to continue for 1 hour at 37° C. to permit adsorption of
virus to the cells. After 1 hour, the compounds of the present inventions
were added at a 1:10 dilution directly to the infected cells. Infection
was allowed to continue for another 16 hours. After this period the media
was removed and the cells fixed and stained. Actin protein was visualized
with fluor-conjugated phalloidin and DNA (viral and cellular) was
visualized by staining with DAPI, as described (see Reeves et al., 2005,
Nat. Med. 11: 731-738). Cells were imaged on a multiwavelength
fluorescence microscope for the presence of cytopathic effect, viral
infection and actin protein tail formation.

[0052] The purpose of the plaque assays is to screen compounds for their
effect on vaccinia virus plaque size, and on the formation of "comet"
plaques, an archipelago of smaller plaques that form adjacent to a large
plaque. Large plaques form as virus from an infected cell egresses, by
means of actin protein tails, and infects an apposing cell. An infected
cell eventually dies leaving a hole in the monolayer. Comet plaques occur
when a form of the virus (called EEV) is released into the supernatant
and settles adjacent to a large plaque. Comets are generally smaller than
large plaques because the initial infection is derived from virus
produced by an adjacent large plaque, not by the initial inoculum. To a
small extent, the size of the large plaques is determined by EEV as well.
Formation of actin protein tails (and thus the size of large plaques)
depends on Src- and Abl-family kinases (Reeves et al., 2005, Nature
Medicine. 11: 731-738), whereas the formation of EEV (and hence comets)
depends on Abl-family kinases. Inhibitors of Abl- and Src-family kinases
result in "pinpoint" plaques (e.g. PD166326), whereas inhibitors of
Abl-family kinases cause somewhat reduced plaque size and loss of comets
(e.g. Gleevec® or STI-571). The Src and Abl family tyrosine kinases
have been found to participate in vaccine virus (VV) action motility and
release of infectious virions, and inhibitors of these tyrosine kinases
block formation of action tails. See WO 2205/072826, the entire
publication is incorporated by reference herein.

[0053] To do the plaque assay, cultured BSC40 cells were added to 12-well
tissue culture dishes at a high density. These cells were allowed to
adhere overnight and reach confluency. The media covering the monolayers
was removed and replaced with low serum media (2% FBS). Approximately
1×103 PFU of vaccinia virus was added to the monolayers and
allowed to adsorb to the cells for 1 hour. Following adsorption, the low
serum media was removed and replaced with complete media (10% FBS).
Compounds of the present invention were added to complete media for a
final concentration of 100 μM. Monolayers were allowed to incubate for
approximately 3 days at 37° C. undisturbed. After this period, the
media is removed and cells are fixed and stained with a Crystal Violet
solution, and scored for plaque size or the presence of comets.

[0054] Compounds as disclosed in Summary Table B (See Table B) have been
identified that have activity against poxvirus and specifically vaccinia
virus (VV) based on the plaque assays. For instance, FIG. 1 shows
compounds Eph--2wbz--105, Eph--2wbz--203,
Eph--2wbz--206 and LG2-71 produce small plaques with comets
(FIG. 1B), whereas compounds DM-1-187 and DM-1-196 produce smaller
(pinpoint) plaques with no comets (FIG. 1C). Compounds
Eph--2wbz--110, Apck108, Apck111, Apck26, and Apck27 produce
pinpoint plaques (FIG. 2A), whereas compounds Apck105, LG2-91 and LG2-96
produce no plaques (FIG. 2B). Moreover, FIG. 4 illustrates additional
phenotypes: such as small plaques with large comets produced by compounds
Apck34 and Apck32 (FIG. 4A); more plaques than WT were produced by
treated with compounds JGAP-13 and Butyeolactones-1 (FIG. 4B); and
damaged monolayer was produced by treated with compounds Apck101 and
YYB21 (FIG. 4C).

[0056] Some of the compounds tested herewith, e.g., ApCK103, Apck-43,
LG2-55, and LG2-71 had effects in both the Herpes and Vaccinia assays
(see also below, and Table B below). Others (e.g. PD166326 and related
compounds described in previous applications) had effects in both
vaccinia assays and assays with pathogenic E. coli (Swimm et al., 2004,
Molecular Biology of the Cell. 2004. 15:3520-3529). Some of the Class II
and III compounds were also tested in microscopy assays as described
above. The results showed that Class II compounds tested in that assay
did not affect the number of actin tails, whereas Class III compounds
tested in that assay reduced or eliminated actin tails (See FIG. 3). As
described above, FIG. 3 illustrates actin protein tail and plaque
formations from microscopy and plaque vaccinia assays for wide type (WT,
with only the vaccinia virus infection) (top row) and with compounds
STI-F (middle row) and Eph--2wbz--203 (bottom row), and their
likely kinase family targets. Based on the characterization of the
kinase-dependence of actin motility, these data indicate that Class II
compounds likely inhibit Abl-family kinases and Class III compounds
likely inhibit both Abl- and Src-family kinases, though there might be a
possibility that other kinases are also inhibited.

[0057] The results provided herewith also provide implications for a
treatment of poxyiral infections. Because the phenotypes caused by
Gleevec®, an inhibitor used for the treatment of poxyiral infections,
are consistent with the phenotypes caused by the Classes II and III
compounds described herewith, it suggests that both Class II and Class
III compounds will likely block EEV release. Because EEV mediate
dissemination of the virus in vivo, these compounds will likely confine
the infection to a particular locale (e.g. lungs). Furthermore, because
Gleevec® does not interfere with the acquisition of protective
immunity, immunosuppressive effects of the Class II or Class III
compounds provided herewith would not be expected.

Example 3

Drug Screening Assays for Herpes Virus

[0058] All herpes viruses share the property of establishing life-long
infection in their host. Notably, the gamma-herpes viruses are all
associated with the development of lymphomas and other cancers. To
determine whether tyrosine kinases participate in gamma-herpes virus
infections, confluent monolayers of 3T3 cells were exposed and plated in
optical 96 well dishes to the library of compounds of the present
invention described herein for 1 hour. The cells were then infected with
a gamma-herpes variant that expresses GFP under a CMV promoter
(GHV-Bac-GFP), and replaced the compounds of the present invention at
final concentration of 10 μM.

[0059] After 7 days, control cells that were left untreated exhibited
marked cytopathic effects, an effect attributed to the spread of the
initial infection throughout the monolayer, and subsequent lysis of
infected cells. Amongst compound treated cells, three phenotypes were
evident: (i) compound treated cells showed evidence of cytopathic effects
to the same extent as controls. Because compound treated cells left
uninfected showed little evidence of cytopathic effects this phenotype
indicates that compounds causing this phenotype did not affect viral
entry, egress from an infected cell, spread within the monolayer, or
lysis; (ii) Monolayers of cells remained intact after treated with this
group of compounds, and examination of the GFP fluorescence indicated
foci of fluorescence that did not spread throughout the monolayer. This
phenotype indicates that the compounds causing this phenotype likely
block virus entry or egress. Exemplary compounds include, but are not
limited to CGP-2 (Gleevec®), StiAF3-iAR, and LG2-71 compounds of the
present invention (See Table B below); and (iii) Monolayers of cells also
remained intact after treated with this group of compounds, but
examination of the GFP fluorescence indicated fluorescence throughout the
monolayer. This phenotype indicates that the compounds causing this
phenotype did not block viral entry or egress but may inhibit cellular
lysis. Exemplary compounds include, but are not limited to CGP51148WBZ-4,
Apck103, Apck21, APck25, APcK36, ApcK 42, APCK50, APCK51, APCK53, LG2-55,
LG2-77, and LG2-81 (See Table B below). Together these data suggest that
compounds in groups (ii) and (iii) affect aspects of viral growth, and
limit production of new virus, and are further expected to be useful for
treating and preventing pathogenic infections.

[0060] Although these compounds provided herewith are designed to inhibit
tyrosine kinases, there is no evidence in the literature for the
involvement of tyrosine kinases in gamma Herpes pathogenesis, and
off-site effects of these compounds on cellular or viral targets would
not be ruled out. Nevertheless, the compounds identified herewith may
prove effective in treating infections caused by Herpes virus and related
virus, including, but not limited to Epstein Barr virus and Herpes
Simplex virus.

[0061] Many modifications and other embodiments of the inventions set
forth herein will come to mind to one skilled in the art to which these
inventions pertain having the benefit of the teachings presented in the
foregoing descriptions and the associated drawings. Therefore, it is to
be understood that the inventions are not be limited to the specific
embodiments disclosed and that modifications and other embodiments are
intended to be included within the scope of the appended claims. Although
specific terms are employed herein, they are used in a generic and
descriptive sense only and not for purpose of limitation. Further, it
must be noted that as used in this specification and the appended
embodiments, the singular forms "a," "an," and "the" include plural
references unless the context clearly dictates otherwise.

[0062] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the art to
which this invention pertains. All publications and patent applications
are herein incorporated by reference to the same extent as if each
individual publication or patent application was specifically and
individually indicated to be incorporated by reference.